modified leishman stain: the mystery unfolds...leishman stain which is widely used in diagnostic...

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*Corresponding author: Email: [email protected];

British Journal of Medicine & Medical Research4(27): 4591-4606, 2014

SCIENCEDOMAIN internationalwww.sciencedomain.org

Modified Leishman Stain: The Mystery Unfolds

K. A. Fasakin1*, G. R. A. Okogun2, C. T. Omisakin1, A. A. Adeyemi3and A. J. Esan1

1Department of Haematology, Federal Medical Centre, P.M.B 201, Ido Ekiti, Nigeria.2Department of Medical Laboratory Science, Ambrose Alli University, P.M.B 114, Ekpoma,

Nigeria.3Department of Chemical Pathology, Obafemi Awolowo University Teaching Hospitals

Complex, P.M.B 5538, Ile-Ife, Nigeria.

Authors’ contributions

This work was carried out in collaboration between all authors. Author KAF designed thestudy, performed the statistical analysis, wrote the protocol, and wrote the first draft of the

manuscript. Author GRAO was also involved in study design and assessment of theliterature. Authors CTO and AAA managed the analyses of the study. Authors KAF and AJE

managed the literature searches. All authors read and approved the final manuscript.

Received 13th April 2014Accepted 21st May 2014

Published 19th June 2014

ABSTRACT

Background: Current Leishman staining technique for staining thin blood films fordifferential leukocyte count is too time consuming to meet emergency needs inhospitalized patients with infectious and other deadly diseases. This study aimed atdiscovering optimal phenol: Leishman powder ratio appropriate for modified Leishmanstain and finding an optimized staining reaction and facilitating rapid cellular analysis ofblood without alteration in quantity and quality.Methodology: Leishman stain was modified using phenol crystals and liquefied phenol.Various ratios of phenol and Leishman powder were experimented in absolute methanol.Fixing and staining times of staining process were manipulated to develop new stainingprocedures that gave optimal staining reaction on thin blood films prepared within twohours of receipt. Results were presented as photomicrographs of stained slides.Results: 30mg and 50mg of phenol crystals or 30µL and 50µL of liquefied phenol wererequired to give 1:5 and 1:3 phenol: Leishman powder ratios respectively. Two modifiedLeishman staining techniques were developed. The first fixed thin blood films for 25seconds and stained for 50 seconds while the second technique fixed slides for 1 minute

Original Research ArticleRRRreResearch……..

Article

British Journal of Medicine & Medical Research, 4(27): 4591-4606, 2014

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and stained for 3 minutes. Photomicrographs of thin blood films showed excellent stainingresults that compared well with the conventional technique.Conclusion: Unlike the conventional method which requires a total of 10-12 minutes, tocomplete the staining process, modified Leishman staining techniques require only 75.0seconds and 4.0 minutes! Batches of blood films can be stained within a short time thusfacilitating rapid diagnosis and treatment of patients.

Keywords: Phenol; accentuating agent; Romanowsky; azure b; modified Leishman stain;photomicrograph; thin film; staining time.

ABBREVIATIONS

WBC: White blood cell; RBC: Red blood cell; DMSO: Dimethyl sulphoxide;Polymorphonuclear cells: Cells having two or more nucleus separated by mitotic strands.While the term generally describes neutrophils, eosinophils and basophils it is sometimesused to refer to neutrophils; Mononuclear: Having one nucleus; pH: This is a measure ofhydrogen ion concentration; a measure of the acidity or alkalinity of a solution. Aqueoussolutions at 25ºC with a pH less than seven are acidic, while those with a pH greater thanseven are basic or alkaline. A pH level of 7.0 at 25ºC is defined as 'neutral' because theconcentration of H3O

+ equals the concentration of OH− in pure water; mg: milligram; µL:Microlitre

1. INTRODUCTION

The need for rapid testing and diagnosis of diseases has posed great challenges to us.Leishman stain which is widely used in diagnostic haematology laboratory has not enjoyedscientific touch for some decades as other Romanowsky dyes except in the work ofWoronzoff-dashkoff where it constituted one of the several components that made up thecomplex dye [1]. Besides, to the best of our knowledge there is lack of literature on themodification of Leishman stain using accentuating agents. This study introducesaccentuating agent phenol as the ‘vehicle of innovation’, manipulation of phenol: Leishmanpowder ratios as the ‘steering wheel’ and development of modified Leishman stainingtechniques as the ‘fuel’ to rapid testing and diagnosis of diseases by Leishman stainingmethods.

Romanowsky dyes are widely used in the diagnostic laboratory and Leishman stain occupiesan intermediate position among the various modifications of Romanowsky dyes available.Generally, Romanowsky dyes are composed of combinations of acid and basic dyes and areuniversally employed for routine staining of blood films [2]. The remarkable property of theRomanowsky stains of making subtle distinctions in shades of staining granules differentiallydepends on two components, namely, azure B (trimethylthionine) and eosin Y(tetrabromofluorescein) [3-4]. Azure B is one of the oxidative products of methylene blue andis superior to other azures, the reason it is more commonly employed in Romanowsky dyes[5]. The original Romanowsky combination was polychrome methylene blue and eosin. Theazure B and eosin Y was advocated for the International Committee for Standardization inHaematology (ICSH) [6]. Lapen and Marshall and their co-researchers described newpolychrome stains which are composed of measured amount of pure azure B and eosin Y[7-8]. According to Bins et al, the preparations have the advantage of being standardizedbecause oxidation is complete in these stains and extraneous elements are eliminated [9].


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Thus, it ensures consistent results from batch to batch. The stability appeared to vary and isdependent on the concentration of the dyes, the molarity of the buffer solutions and thepresence of dimethyl sulphoxide (DMSO) as a stabilizer. ‘Although most staining solutionsincluding the routine May Grünwald-Giemsa stain showed marked loss of staining capacitysoon after preparation’, it was possible to obtain an azure B–eosin Y mixture with verysatisfactory staining properties which did not decrease during 8 h after its preparation, saidBins and his colleagues. Leishman stain and Wright stain have similar staining results. Thelatter is widely used in North America. Newer modifications of Romanowsky dyes whichgive superior staining of thin and thick smears have been developed. These areRomanowsky-Giemsa dyes, namely, May-Grünwald-Giemsa (MGG), Jenner-Giemsa andWright-Giemsa. Modifications differ in the ratios of dye components and manufacturingmethods used [10-15]. They require preparations of buffers at different pH to ensure properstaining else staining becomes inadequate. More complex procedures are also involvedwhich require strict monitoring. Alone, Giemsa stain is inadequate for staining red cells,platelets and leukocyte cytoplasm and too cumbersome to meet emergency needs althoughbatches of films can be stained [16]. In the recent times, Begaumi and Shetty describedLeishman-Giemsa cocktail as a new and potentially useful cytological technique comparableto Papaniculaou staining for oral cancer diagnosis but has not been experimented forstaining blood cells in peripheral blood films [17]. According to the researchers, itsapplication requires further study. Marthur and his colleagues recently attempted using ascalable system to classify white blood cells after staining with Leishman stain for differentialleukocyte count [18]. The goal of differential leukocyte count is to provide valuable clinicalinformation in health and disease [19]. Differential leukocyte count involves enumeration ofpolymorphonuclear cells (neutrophils, eosinophils and basophils) and mononuclear cells(monocytes and lymphocytes) in peripheral blood film by manual microscopic approach. Onehundred leukocytes must be counted and quantitative and qualitative abnormalities observedin any of the leukocytes help in clinical diagnosis. The most commonly used method is thebattlement technique. Generally, it is performed as part of complete blood count analysiswhich involves quantitative and qualitative evaluation of red blood cells (RBC), white bloodcells (WBC) and platelets (Plt) in a whole blood.

Leishman stain is the stain of choice in this part of the world for the staining of blood cells fordifferential leukocyte count analysis. Conventional preparations of 100ml of Leishmanstaining solution requires dissolution of 150mg of Leishman powder in 100ml of absolutemethanol, warm up to 50ºC, and shake gently at intervals to aid dissolution. It is then filteredbefore use. Recently, Bain and his colleagues decried use of 200mg instead of the usual150mg for optimal staining [20]. Modified stain seeks to optimize staining of cellularelements of the blood by a completely different approach. This method uses theaccentuating property of phenol to optimize staining. Phenol was first used as one of thecomponent dyes in Ziehl Neelson stain where it acted as an accentuating agent [21]. Wehypothesized that if phenol optimized staining in Ziehl Neelson procedure, it should do thesame in Leishman stain given the stirring fact that phenol and methyl alcohol have terminalhydroxyl functional groups (-0H) which expectedly should aid their reactivity. Besides, sincepart of our focus is enhancing timely generation of results, we assume that phenol throughits contribution to ripening of the modified Leishman staining solution should shorten fixingand staining times of newly developed procedures. This should in turn contribute to overallreduction in turn-around time (TAT) of differential leukocyte counts test using properlystained thin blood films.


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2. ACCENTUATING AGENTS

These are chemical substances that are used to improve the quality of staining of tissue(fluid tissue inclusive). Without them staining can take place but it will be of low quality. Theyact by changing the pH of the staining solution with overall effect of increasing the rate ofstaining uptake by the tissue [21].

2.1 Phenol: The Modifier

The main active ingredient responsible for modification in this study is phenol. Phenol alsoknown as phenyl alcohol is a polar organic compound with an aromatic ring and hydroxylfunctional group. It has a chemical formula C6H5OH [22]. In the crystal form it appears as aglittering white solid but in liquefied form as colorless liquid (with aromatic smell) except athigh temperature when it turns pinkish following oxidation. It has pKa of approximately 10(9∙95) and it is more acidic than its alcoholic counterpart (cyclohexanol) with pKa of 16 butless acidic than alkanoic acids. Several articles have helped to explain the increased acidityof phenol over its alcoholic counterparts [23-26].

2.2 Aim of the Study

This study aimed at discovering optimal phenol: Leishman powder ratio appropriate formodified Leishman stain and finding an optimized staining reaction and facilitating rapidcellular analysis of blood without alteration in quantity and quality thus facilitating timelygeneration of results which enhance early diagnosis.

3. METHODOLOGY

3.1 Study Location

This study was carried out at the haematology department of Federal Medical Centre, IdoEkiti. Extra thin blood films were made from blood samples of patients who came for routinefull blood count analysis following an informed consent as part of the ethical guidelines.

3.2 Preparation of Blood Film for Examination

The manual wedge method described by Jean Safer was used in blood film preparation [5].

3.3 Components of Modified Leishman Stain

Component ingredients of modified Leishman stain are Leishman powder, absolutemethanol, phenol crystals or liquefied phenol

3.4 Determination of Phenol: Leishman Powder Ratio/100ml of LeishmanStaining Solution Using Phenol Crystals

We weighed 30mg, 37∙5mg, 50mg, 75mg and 150mg of phenol crystals alongside with150mg of commercially-prepared Leishman powder into different brown screw-cappedbottles to give phenol: Leishman powder ratios 1:5, 1:4, 1:3, 1:2 and 1:1 respectively anddissolved in 100ml of absolute methanol to make up 100ml of modified Leishman solution.


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The preparation was done at room temperature (18-25ºC) and the stain was kept away fromdirect sunlight. The protocol is shown in Table 1.

Table 1. Protocol of Preparing Modified Leishman Staining Solution with PhenolCrystals

Slides 1 2 3 4 5Phenol (mg) 30.0 37.5 50.0 75.0 150.0Leishman powder (mg) 150.0 150.0 150.0 150.0 150.0Phenol: Leishman powder Ratio 1:5 1:4 1:3 1:2 1:1Volume of absolute methanol (ml) 100.0 100.0 100.0 100.0 100.0Assessment of thin blood filmsafter staining with modifiedLeishman stain

DistinctlyStained

FairlyStained

DistinctlyStained

PoorlyStained

BadlyStained

Slides 1 and 3 became the reference slides. 1:5 and 1:3 became the reference phenol: Leishmanpowder ratios with optimal staining performance

3.5 Determination of Phenol: Leishman Powder Ratio/100ml of LeishmanStaining Solution Using Liquefied Phenol

We dissolved 30µL, 37∙5µL, 50µL, 75µL and 150µL each of liquefied phenol in 100ml ofabsolute methanol. The resultant phenol-methanol mixture was used to dissolve 150mg ofcommercially-prepared Leishman powder already weighed into different brown screw-capped bottles to give phenol: Leishman powder ratios 1:5, 1:4, 1:3, 1:2 and 1:1 respectivelyin 100ml of staining solution. The preparation was done at room temperature (18-25ᴼC) andthe stain was kept away from direct sunlight. The protocol is shown in Table 2. Thealternative method followed the conventional method of Leishman stain preparation [16].Following the Leishman stain preparation, the phenols were added to the staining solution togive phenol: Leishman powder ratios 1:5, 1:4, 1:3, 1:2 and 1:1 respectively in 100ml ofstaining solution.

Table 2. Protocol of preparing modified leishman staining solution with liquid phenol

Slides 1 2 3 4 5Phenol (µL) 30.0 37.5 50.0 75.0 150.0Leishman powder (mg) 150.0 150.0 150.0 150.0 150.0Phenol: Leishman powder Ratio 1:5 1:4 1:3 1:2 1:1Volume of absolute methanol (ml) 100.0 100.0 100.0 100.0 100.0Assessment of thin blood films afterstaining with modified Leishman stain

DistinctlyStained

FairlyStained

DistinctlyStained

PoorlyStained

BadlyStained

Slides 1 and 3 became the reference slides. 1:5 and 1:3 became the reference phenol: Leishman powderratios with optimal staining performance

3.6 Determination of the Fixing and Staining Times of Staining Process

Inappropriate fixing and staining times of staining process often result in under-staining orover-staining of thin blood films. This often causes erroneous differential count results.Accurate diagnosis of diseases either infectious, haematological, biochemical, metabolic,systemic or otherwise through complete blood count analysis is partly is based on properlystained thin blood films for differential leukocyte count.


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The fixing and staining times of staining process were determined by manipulating theexperimental times thus leading to development of new staining procedures. Two categoriesof fixing and staining times were experimented.

3.6.1 Category 1

Seven thin blood films from enrolled patients were made and labelled 1-7. The fixing andstaining times were measured in seconds. The specific times that gave the optimal stainingreaction constituted the fixing and staining times of the first technique. This technique wasdeveloped for extreme emergency situation Table 3 showed the protocol.

3.6.2 Category 2

Five thin blood films from enrolled patients were made and labelled 1-5. The fixing andstaining times were measured in minutes. The specific times that give the optimal stainingreaction constitute the fixing and staining times of the second technique. Table 4 showed theprotocol.

Table 3. Protocol showing Fixing and Staining times of Modified Leishman StainingTechnique

Slides 1 2 3 4 5 6 7Fixing time(seconds)

5.0 7.5 10.0 15.0 20.0 25.0 30.0

Staining time(seconds)

10.0 15.0 20.0 30.0 40.0 50.0 60.0

Stainingevaluation

Poorlystained

Poorlystained

Poorlystained

Poorlystained

ModeratelyStained

Goodstaining

Moderatelystained

Slide 6 was the reference slide. All cellular elements of the blood were satisfactorily stained

Table 4. Protocol showing Fixing and Staining times of Modified Leishman StainingTechnique

Slides 1 2 3 4 5Fixing time (minutes) 1.0 1.0 1.5 1.5 2.0Staining time (minutes) 2.0 3.0 2.0 3.0 2.0Staining evaluation Poorly

stainedExcellentlystained

Poorlystained

Moderatelystained

Poorlystained

Slide 2 was the reference slide. All cellular elements of the blood were excellently stained

4. RESULTS

Research findings revealed that modified Leishman stain can be prepared at two appropriatephenol: Leishman powder ratios 1:5 and 1:3 for both phenol crystals and liquefied phenol.These led to two methods of preparation for the modified Leishman stain.


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4.1 Experimental Outcomes for the Preparation of 100ml Modified LeishmanStain Using Liquefied Phenol

100ml of Modified Leishman stain can be prepared in two ways:

a. Weigh 150mg of Leishman powder into a brown container. Dissolve 50µL or 30 µLof phenol in 100ml of acetone-free absolute methanol depending on the desiredphenol: Leishman powder ratio. The resultant methanol-phenol mixture shouldthereafter be used to dissolve the Leishman powder in a step-wise manner. Theresulting modified Leishman staining solution must be filtered into a brown screw-capped bottle and can be used immediately or kept overnight for further ripening.

b. This second approach follows the conventional method of Leishman stainpreparation [16]. Weigh 150mg of Leishman powder into a brown container and add100ml of absolute methanol in a stepwise manner until all the stain has dissolved.Depending on the desired ratio, add 50µL or 30µL of phenol to the prepared stain.

It is worth noting that more volume can be prepared but then phenol: Leishman powder ratiomust be put into consideration and the preparation must be done at room temperature.

This research work has led to the development of two modified Leishman stainingtechniques. The first technique stained thin blood films for overall time of 75 seconds whilethe second stained thin blood films for a total of 4∙0 minutes. Sörenson’s buffer or tap waterof pH 6∙8 was used for dilution in such a way that the volume of buffer or tap water doublesthat of stain during the staining procedure.

4.2 Experimental Outcome Describing Modified Leishman StainingTechnique 1

Blood film was made using a scrupulously clean grease-free glass slide and air-dried. Thefilm was flooded with modified Leishman stain for exactly 25 seconds. At exactly 25 secondsSörenson’s buffer or tap water of pH 6.8 was added in such a way that the volume of bufferor tap water doubles that of stain. It was left for 50 seconds. At exactly 50 seconds, the slidewas rinsed with buffer or tap water carefully so that no stain remains on the slide. The backof the stained blood film was cleaned with dry swab to remove any residual stain at the backof the slide. The stained blood film was air-dried and made to stand upright on a slide rack. Itwas examined with oil immersion objective for differential leukocyte count and filmappearance.

4.3 Experimental Outcome Describing Modified Leishman StainingTechnique 2

Blood film was made using a scrupulously clean grease-free glass slide and air-dried. Thefilm was flooded with modified Leishman stain for exactly 1 minute. At exactly one minute,Sörenson’s buffer or tap water of pH 6.8 was added in such a way that the volume of bufferor tap water doubles that of stain. It was left for three minutes. At exactly three minutes, theslide was rinsed with buffer or tap water carefully so that no stain remains on the slide. Theback of the stained film was cleaned with dry swab to remove any residual stain at the backof the slide. The stained blood film was allowed to stand upright and air-dry on a slide rack. Itwas examined with oil immersion objective for differential leukocyte count and filmappearance.


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Comparative study of the first and second modified Leishman staining techniques revealedclearer morphologic pictures with the latter. Results are presented as photomicrographs ofstained slides as represented by Figs. 1A and B – 3A, B and C.

Fig. 1A. Photomicrograph of Thin blood film showing a Monocyte and normocyticnormochromic RBCs stained with Unmodified (Conventional) Leishman stain (x1000

magnification)

Fig. 1B. Photomicrograph of Thin blood film showing normal Lymphocyte (upperright), giant platelet (lower left) and Normocytic normochromic RBCs stained with

Modified Leishman stain, 4 minutes (x1000 magnification)


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Fig. 2A. Photomicrograph of stained thin Blood films showing normocytic,normochromicRBCs, segmented Neutrophil and Eosinophil stained with Modified

Leishman stain, 4 minutes X 1000 magnification (Phenol-Methanol method ofPreparation)

Fig. 2B. Photomicrographs of stained thin Blood films showing normocytic,Normochromic RBCs, Eosinophil stained with Modified Leishman stain, 4 minutes

X 1000 magnification (Conventional method of Preparation)


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Fig. 3A. Photomicrograph of Thin blood film showing EosinophilAnd Normocytic normochromic RBCs stained with Modified Leishman

Stain, 75seconds (X1000 magnification)

Fig. 3B. Photomicrograph of blood film showing lymphocyte and normocyticnormochromic RBCs Modified Leishman stain, 75 seconds (X 1000 magnification)


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Fig. 3C. Photomicrograph of Thin blood film showing normal Lymphocyte andNormocytic normochromic RBCs stained with Modified Leishman stain, 4 Minutes

(x1000 magnification)

4.4 The Mechanism of Action of Modified Leishman Staining Techniques

Phenol alters the pH of the modified Leishman stain thus increasing its permeability by theformed elements of the fluid tissue. This alteration in pH in turn shortened the overall timerequired for staining. Complete dissolution of phenol in the modified Leishman stain resultedfrom its high solubility and reactivity with absolute methanol. Both phenol and methanol arepolar, organic compounds with terminal hydroxyl functional groups which facilitate theirreactivity. Phenol turns blue litmus paper to red. Although the pH of the modified stain couldnot be determined due to possible impartation of colour on the sensitive membrane of theglass electrode of the pH meter, there is evidence that modified Leishman stain is slightlymore acidic than its conventional counterpart. While terminal hydroxyl (-OH) functionalgroups of phenol and methyl alcohol aids their reactivity, the benzene ring of phenol acts aselectron withdrawing group.

4.5 Principle of Modified Leishman Staining Technique

A blood film review is first performed on an appropriately prepared and stained blood filmusing modified Leishman staining technique. This involves a systematic microscopic scan toestimate leukocyte count and classify them into group types, identify morphologicalabnormalities of erythrocyte and estimate platelet number.

5. DISCUSSION

Romanowsky stains have gained popularity over several decades in the field of haematologyand other disciplines in clinical laboratory sciences. In 1944, Field’s stain was described as arapid technique for the staining of thick films for malaria parasites but has been modified togive fairly satisfactory results for staining thin blood films [27]. Its universal application indiagnostic haematology laboratory was handicapped by the short staining intervals between


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steps which make its use impractical in larger laboratories. Simeon’s modification of Boye’sand Sterenel’s method has been developed [28]. The method is only useful when methylalcohol is unavailable to prepare Leishman stain. Its nine stepwise procedures in quicksuccessions make it unpopular and impractical in routine staining of blood films fordifferential leukocyte count and peripheral blood film examination. Researchers further havesought to perform differential counts by automation but these are not without their limitations.Flow cytometry is the reference method and the most popular among them [29-31]. Cost,need for calibrations and re-calibrations of equipment, availability of reagents andconsumables, trained personnel, maintenance and technicalities of these equipment andaccessibility of test to patients in resource-limited setting are some of the challengesobserved with automated analyzers.

The discovery of the accentuating benefit of phenol in modifying Leishman stain has led to amore practical and rapid method of staining thin blood films for routine complete blood countanalyses and research purposes. This stain, in practice, is believed to find its majorcontribution in reducing morbidity and mortality due to infections and other deadly diseasesin extreme emergency cases where timely generation of results by rapid techniquesbecomes highly imperative. Figs. 1 A and 1B, 2A and 2B, 3A, 3B and 3C showed theoutcomes of our research. As it can be observed from the photomicrographs of stained thinblood films 1A and 1B, the second modified Leishman staining technique, which required anoverall 4 minutes for staining compared well with the conventional, unmodified method ofstaining technique as it stained all the cellular elements of the blood as the control method.The observed blurred outline of the monocyte in Fig. 1A stained with the conventionalmethod is due to manual method of photomicrography and not the stain. This we inferredowing to similar observation in some of the blood pictures of thin blood films stained with themodified Leishman stain. Where images are taken with improved photomicrographytechnique such as the use of digital microscope connected with computer system, the clarityof the morphologic pictures of blood cells will stand out more clearly. In addition, Fig. 2Ashowed the excellent staining characteristics and pre-eminence of modified Leishmanstaining solution prepared by dissolving the phenol component in acetone-free absolutemethanol and in turn use the resultant phenol-methanol mixture to dissolve the Leishmanpowder in a step-wise manner (i.e. 1st method of preparing modified Leishman stain usingliquefied phenol) over the conventional method of preparation (i.e. 2nd method).Theleukocytes, platelets and red blood cells stained more distinctly in phenol-methanol mixturemethod than the conventional method. The fine pinkish neutrophil granules could be seen asdistinct from the orange red granules characteristic of eosinophils. The conventional methodof modified Leishman staining preparation when used irrespective of staining techniquesgave morphologic pictures of slightly lower quality than when compared to phenol-methanolmethod. The mechanism behind this still remains unclear but we assume that thebiochemical interaction that existed between phenol and methanol before coming intocontact with Leishman powder was responsible for this. To further elucidate our scientificfindings, Figs. 3A and 3B showed the photomicrographs of thin blood films stained for overallof 75 seconds, and 3C showed that stained for overall time of 4 minutes. It was clearlyobserved that the latter modified Leishman staining procedure, more often than not, gavebetter staining characteristics and morphological pictures of blood cells in peripheral bloodfilms than the former. The observed incomplete staining of the eosinophilic granules was dueto low staining uptake by the nucleus of the white blood cell due to shorter fixing time. Aproperly fixed smear will have all the components of the blood cells appropriately fixed withno experience of blood film washing off during staining process. However, these results stillrevealed that staining thin blood films for differential leukocyte counts within seconds to fewminutes is a great possibility especially in extreme emergency cases. We based all the


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aforementioned assessments regarding the staining quality of the dye on blood films on theconsistency of the staining uptake by leukocytes, red blood cells and platelets during fixingstage from slide to slide, non-deposition of artifacts, no quantitative loss due to non-identification or mis-identification of blood cells, and already established appearance of redblood cells (salmon pink) leukocytes including the nuclei (purple) and cytoplasmic granules(specific for each granulocytic leukocyte), and platelets (purple). When stained thin bloodfilms were assessed by eight different researchers and scientists, the modified stainingtechnique demonstrated these good qualities especially when staining procedure wasperformed using the 4 minutes procedure (i.e. the second staining technique) more than the75 seconds procedure. A properly stained thin film when appropriately stored can keep for 6months to 1 year.

Many factors have been identified which interfere with staining quality. Most of the factorsaffect both conventional and modified Leishman stain alike. Among the factors include non-absoluteness of methyl alcohol. Absolute methanol with ≤ 1% water gave excellent stainingresults while methanol with ≥5% water introduces artifacts into the blood film. The degree ofartifacts depends on the degree of deviation from the absoluteness of this solvent. Bothrefrigerated and high temperature affect the quality of staining but the effect is more on themodified Leishman stain as high temperature of close to 46ºC induces oxidation of phenoland shortens the shelf-life of the stain. Other factors that affect the quality of staining of bothconventional Leishman stain and modified Leishman stain include non-performance of testaccording to standard operating procedures, using buffer or tap water of incorrect pH. Theeffect of using buffer of incorrect pH is more on the quality of staining produced by modifiedLeishman stain than the conventional counterpart. This includes but not limited to non-staining of the basophilic or acidophilic features of leukocytes distinctly. Our researchfindings did not differ from what Nguyen, Jean et al. and Dacie and his group presented oncauses of faulty staining [5,16,32].

Of significant importance is our finding on the effect of using powdered latex gloves whilestaining. The use of powdered latex gloves creates appearance of tiny droplets on blood filmas can be observed in Figs. 1A, 3A and 3C which affect absolute clarity of morphologicpictures. The more powdered the latex gloves the more the degree of appearance of tinydroplets of artefactual deposits on the blood film. Comparing the morphologic appearance ofeosinophils in Figs. 2A, 2B and 3A, it is very clear that the orange-red colour whichcharacterizes Eosinophil’s granules stands out more clearly with the second procedure thanthe first.

Again, our research presents a novel and unprecedented findings. The only factor thatselectively affects modified Leishman stain is the phenol: Leishman powder ratio. Excessphenol: Leishman powder ratio results in slides turning blue-black almost immediately due tosharp changes in the overall pH of the modified Leishman stain and attendant extremely lowfixing and staining times of staining process. On the other hand, lower phenol: Leishmanpowder ratio results in failure of the modified Leishman stain to fix and appropriately stainnuclei of leukocytes. We also noted that phenol created some buffering effect. Whilesometimes, staining for overall of 12 minutes when using conventional Leishman stain mightbe impossible (in fact, the overall staining time might have to be increased to roughly 15-17minutes), we still stained with modified Leishman stain at the exact fixing and staining timeswith good to excellent staining characteristics of blood cells depending on the method ofpreparation and staining technique used. This we inferred because both conventional andmodified Leishman stain were prepared with the same dye and absolute methanol except for


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the addition of phenol in the modified method. We assume phenol’s alteration of the overallpH of modified Leishman staining solution may be responsible.

6. PRECAUTIONS

The stain should not be refrigerated or kept above room temperature for optimal stainingquality. Highly purified and acetone-free absolute methanol must also be used andpowdered-free gloves are encouraged. Phenol should be handled with care with limitedexposure time owing to its established toxicity. The Scientist should be on necessaryprotective wares during preparation.

7. CONCLUSION

Modified Leishman stain and its applications will significantly affect the practice of diagnostichaematology. This inference is based on the simplicity and practicality of staining techniquesand cost-effectiveness of the modified Leishman stain when compared to the ease of incomegeneration. Besides, the stability of the stain and the understanding of the mechanism ofaction of this novel dye as well as opportunity to have sizeable batches of thin blood filmsstained lend credence to its use in diagnostic haematology laboratories. With the advent ofautomated slide stainer, batches of thin blood films can be stained within 75 seconds and 4minutes in busy private and public laboratories. With the advent of these new rapidtechniques in place, morbidity and mortality which resulted from late haematological findingswill be drastically reduced especially in hospitalized patients with severe illness.

CONSENT

All authors declare that written informed consent was obtained from the patients (or otherapproved parties) for publication of this case report and accompanying images.

ETHICAL APPROVAL

This research work was performed according to ethical guidelines of Federal MedicalCentre, Ido Ekiti following an informed consent to perform routine analysis. No bio-data ofthe patients were required for research.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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© 2014 Fasakin et al.; This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Peer-review history:The peer review history for this paper can be accessed here:

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